There is great interest in manipulating autophagy to improve cancer treatment but considerable confusion about how to do so. For example, it has become clear in the last few years that many anti-cancer treatments induce autophagy in tumor cells, however, because this autophagy has been reported to both protect and kill tumor cells, even such a basic question as whether we should try to increase or decrease autophagy during cancer treatment is unclear. Despite this uncertainty, clinical trials are being developed that combine autophagy inhibitors with other drugs while other patients are treated with drugs that induce autophagy but without any consideration for how this autophagy affects the outcome. Thus a major need in cancer research is to better understand the roles of autophagy in tumor cell death so that we can decide how best to manipulate autophagy in people with cancer. We recently discovered a previously unrecognized function for autophagy: autophagy controls the selective release of the nuclear protein HMGB1 from dying tumor cells. HMGB1 release from dying tumor cells is known to lead to a beneficial tumor-specific immune response through activation of Toll-like Receptors on dendritic cells but may also induce non-beneficial pro-tumorigenic activities. Therefore, our recent findings open up an entirely new issue that needs to be dealt with as we consider how to manipulate autophagy in people. We hypothesize that it is not just the efficiency of tumor cell killing that is important but that the characteristics of the dying tumor cells, exemplified by selective release of HMGB1, also determines the overall success of treatment. And, we propose that autophagy controls both these processes. To test this hypothesis we will use a well-characterized model of metastatic breast cancer to complete the following aims. Aim 1. Determine how autophagy controls the efficiency and characteristics of tumor cell killing by anti-cancer treatments. This aim will test the hypothesis that autophagy regulates both the efficiency of tumor cell killing and the release of HMGB1 from dying cells but does so differently for different kinds of anti-cancer treatments. Aim 2. Determine which aspects of the autophagic process are required for the different responses during tumor cell death. Autophagy is a dynamic process that involves multiple steps; in this aim, we test the hypothesis that different steps in the process are required for the different functions of autophagy and we use novel single cell imaging methods to determine how autophagy regulates the core apoptosis machinery. Aim 3. Determine how manipulation of autophagy alters the long-term efficacy of cancer treatment in vivo. In this aim, we test the hypothesis that autophagy manipulation can determine the long term effectiveness of breast cancer treatment using a model of adjuvant and neoadjuvant chemotherapy and we will determine which functions of autophagy are important for controlling the response to therapy. These studies will give us new insights into the role of autophagy during tumor cell death and should provide a rationale for developing autophagy manipulation strategies to improve the effectiveness of cancer treatment. PUBLIC HEALTH RELEVANCE: In the last few years it has become clear that a hitherto understudied cellular process called autophagy is an important regulator of cancer development and treatment. Although it is widely believed that autophagy is important during tumor cell death (e.g. after treatment with anti-cancer drugs), the precise roles for autophagy are unclear and it has been reported that autophagy may both protect tumor cells and cause their death under different circumstances. Our lack of understanding of what autophagy does during tumor cell death limits our ability to manipulate autophagy in order to improve cancer treatment. We recently discovered a new function for autophagy during tumor cell killing- autophagy regulates the release of a protein that controls the immune system. We propose that this function along with autophagy's ability to control the amount of tumor cell killing will together determine whether efforts to kill cancer cells are effective or not. The work proposed here will test these ideas and determine how autophagy controls the amount and characteristics of tumor cell death and should provide a framework to attempt to manipulate autophagy in people who are being treated for cancer in order to improve the benefits of the treatment.